Fluorescent light detection

ABSTRACT

System and method for fluorescent light detection from biological samples to enhance the numerical aperture and/or reduce the cross-talk of the fluorescent light.

FIELD

The present teachings relate to devices and methods for fluorescentlight detection.

Introduction

Molecular biology and other sciences can utilize fluorescent detectionbecause of its wide acceptance and sensitivity. Fluorescent light can begenerated by exciting dyes in a sample using excitation light orchemical means. The fluorescent light emitted can be diffuse due to lowconcentrations of dye in the sample. It is desirable to collect more ofthe diffuse light to increase the efficiency of fluorescent detection.

Fluorescent light detection systems can benefit from correctiveaberration. It is desirable to enhance the numerical aperture bychanging the material of construction of a numerical aperture (“NA”)enhancing optical element in the fluorescent light detection system suchthat the NA enhancing optical element is constructed of differentmaterial than the housing containing the sample. This can introducespherical aberration into the system. It is desirable to eliminate thespherical aberration by positioning the housing in a source plane thatis offset from the theoretical aplanatic source plane.

Increasing efficiency by collecting more light from fluorescent lightdetection systems can generate cross-talk between different samples. Itis desirable to decrease the cross-talk by separating the lightcollected from different samples.

SUMMARY

According to various embodiments, the present teachings include afluorescent light detection system for analyzing samples including a NAenhancing optical element comprising a truncated sphere, a plurality ofhousings for the samples, and a mask including at least one apertureadapted to reduce cross-talk of fluorescent light from the samples.

According to various embodiments, the present teachings include afluorescent light detection system for analyzing samples including a NAenhancing optical element, a plurality of housings for the samples, amovable mask comprising at least one aperture adapted to reducecross-talk of fluorescent light from the samples, and a translationmechanism for moving the mask.

According to various embodiments, the present teachings include afluorescent light detection system for analyzing samples including a NAenhancing optical element, a plurality of housings for the samples, amask comprising at least one aperture adapted to reduce cross-talk offluorescent light from the samples, wherein the mask is positionedbetween the NA enhancing optical element the plurality of housings, anda translation mechanism for moving the plurality of housings.

According to various embodiments, the present teachings include afluorescent light detection method for analyzing samples includingproviding a plurality of housings for the samples, providing a NAenhancing optical element, providing a mask, and positioning the mask toreduce cross-talk between fluorescent light from the samples.

According to various embodiments, the present teachings include afluorescent light detection system for analyzing samples including a NAenhancing optical element including a truncated sphere and an offset,wherein a source plane of the truncated sphere and a back end of thetruncated sphere bound the offset.

According to various embodiments, the present teachings include afluorescent light detection system for analyzing samples including meansfor collecting light, means for housing the samples, and means forreducing cross-talk of fluorescent light from the samples.

It is to be understood that both the foregoing general description andthe following description of various embodiments are exemplary andexplanatory only and are not restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate various embodiments. In thedrawings,

FIG. 1 illustrates a diagrammatical view of various embodiments of asample, housing, and NA enhancing optical element;

FIGS. 2A-2B illustrate cross-sectional views of various embodiments ofNA enhancing optical elements;

FIGS. 3A-3F and 5 illustrate cross-sectional views of variousembodiments of a NA enhancing optical element;

FIG. 4 illustrate the relationship of the source plane for the NAenhancing optical element and the theoretical aplanatic source plane,where FIG. 4A illustrates the focus of light when the theoreticalaplanatic source plane is used and FIG. 4B illustrates the focus oflight when the source plane according to various teachings of thepresent invention is used.

FIGS. 6A-6B, 7-10 illustrate diagrammatical views of various embodimentsof fluorescent light detection systems;

FIG. 11A illustrates a diagrammatical view of various embodiments of alight detection system;

FIG. 11B illustrates a diagrammatical view of various embodiments of aportion of a detector as illustrated in FIG. 11A;

FIG. 12A illustrates a diagrammatical view of various embodiments of alight detection system;

FIG. 12B illustrates a cross-sectional top view of a portion of variousembodiments of a light detection system as illustrated in FIG. 12A;

FIG. 12C illustrates a cross-sectional side view of a portion of variousembodiments of a light detection system as illustrated in FIG. 12A;

FIGS. 12D-E illustrate diagrammatical views of various embodiments of aportion of a detector as illustrated in FIG. 12A;

FIG. 13 illustrates a diagrammatical view of various embodiments of alight detection system;

FIGS. 14-14A illustrate cross-sectional top views of various embodimentsof a NA enhancing optical element and mask; and

FIGS. 15-16 illustrate cross-sectional side views of various embodimentsof NA enhancing optical element, mask, and housing configurations.

DESCRIPTION OF VARIOUS EMBODIMENTS

Reference will now be made to various exemplary embodiments, examples ofwhich are illustrated in the accompanying drawings. Wherever possible,the same reference numbers are used in the drawings and the descriptionto refer to the same or like parts.

The term “cross-talk” as used herein refers to fluorescent light emittedfrom one sample appearing in the detection position of another sample.The samples can be in different housings or in the same housings. Thecross-talk can be the result of reflection, scattering, and/orrefraction from components in the system.

The term “light source” as used herein refers to a source of irradiance(can be measured in photons/mm²) that can provide excitation thatresults in fluorescent emission. Irradiance can be related tofluorescent light because fluorescent light is proportional to thenumber of photons available from the light source for excitation. Lightsources can include, but are not limited to, lasers, solid state laser,laser diode, diode solid state lasers (DSSL), vertical-cavitysurface-emitting lasers (VCSEL), LEDs, phosphor coated LEDs, organicLEDs, inorganic-organic LEDs, LEDs using quantum dot technology, LEDarrays, filament lamps, arc lamps, gas lamps, and fluorescent tubes.Light sources can have high irradiance, such as lasers, or lowirradiance, such as LEDs.

The term “fluorescent light” as used herein refers to a light emitted byan excited sample. Fluorescent light can be emitted in all directions.Fluorescent light can be related to detection signal because detectionsignal is proportional to the number of photons of fluorescent lightcollected from the sample. Fluorescent light can be emitted by a sampleexcited by excitation light, as in fluorescence, or chemically excited,as in chemiluminescence.

The term “NA enhancing optical element” as used herein refers to asinglet or cemented assembly satisfying at least two aplanaticconditions as illustrated in FIGS. 3A-3E. The aplanatic conditions canreduce the divergence angle of a bundle of fluorescent photons emittedfrom any point in the sample object plane or increasing the convergenceof a bundle of excitation rays delivered to any point in the objectplane. The cemented surfaces can have identical curvature (infinite inthe case of planar surfaces). The uncemented or outside surfaces eachsubstantially satisfy a different aplanatic condition. A NA enhancingoptical element can include a truncated sphere, a spherical surfacecombined with a cylindrical surface, a spherical surface combined with aplanar surface, a meniscus lens, etc. The components of a NA enhancingoptical element can be bonded or coupled with a fluid of suitable indexthat does not substantially fluoresce. The index of the fluid can bematched to material of the lens and/or the material of housing, asopposed to air. The components of a NA enhancing optical element can bestationary or movable relative to each other such as a scanning system.NA enhancing optical elements can be constructed of BK7, PBH71, LaSFN9,or other high index glasses. The term “lens” as used herein refers to asingle component or singlet, such as a truncated sphere, meniscus lens,a concave lens, a convex lens, etc. or a system that can includemultiple components, such as NA enhancing optical elements in FIGS. 3B,3D, 3E, camera lenses, etc.

The term “detector” as used herein refers to any component or system ofcomponents that can detect light including a charged coupled device(CCD), back-side thinned CCD, cooled CCD, a photodiode, a photodiodearray, a photo-multiplier tube (PMT), a PMT array, complimentarymetal-oxide semiconductor (CMOS) sensors, CMOS arrays, acharge-injection device (CID), CID arrays, etc. The term “cycle” as usedherein refers to the period of time that the detector collects lightbefore converting it to electrical signal.

The term “housing” as used herein refers to any structure that providescontainment or support to the sample. The housing can be transparent toprovide entry to excitation light and exit to fluorescent light. Thehousing can be constructed of glass, plastic such as low fluorescenceplastic, fused silica such as synthetic fused silica or syntheticquartz, etc. The housing can take any shape including tubing (varioustypes), capillaries, assemblies of capillaries, etched channel plates,molded channel plates, embossed channel plates, wells in a multi-welltray, chambers in a microcard, regions in a microslide, etc.

The term “dye” as used herein refers to any dye in any form in thesample. The dye can emit fluorescent light via fluorescence orchemiluminescence. Fluorescent dyes can be used to emit differentcolored light depending on the dyes used. Several dyes will be apparentto one skilled in the art of dye chemistry. One or more colors can becollected for each dye to provide identification of the dye or dyesdetected. The dye can be a dye-labeled fragment of nucleotides. The dyecan be a marker triggered by a fragment of nucleotides. The dye can bebased or associated with other chemical species such as proteins,carbohydrates, etc.

The term “translation mechanism” as used herein refers to a mechanismfor moving along at least one axis or path. The translation mechanismcan move the mask, NA enhancing optical element, and/or housings. Thetranslation mechanism can provide controllable movement mechanically(gears, pneumatic, cams, lead screws, ball screws etc.), electrically(actuators, linear motors, etc.), and/or magnetically (induced fieldmovement, solenoids, etc.). The control can be provided by computer orelectrical circuitry designed to provide the desirable movementcorresponding to the detector parameters.

According to various embodiments, the sample can include a dye in afluid or solid. The sample emits fluorescent light in all directions.The collection system collects a portion of this light, typically a coneof light. The NA of the NA enhancing optical element can determine thesize of the cone of light.

According to various embodiments, as illustrated in FIG. 1, sample 10can be bounded by housing 20. Fluorescent light 30 can be refracted toform a cone of light with a half angle 50 available for the NA enhancingoptical element 40. According to Snell's Law, a change in index ofrefraction in the light path can refract the light and thus affect thesize of the cone and therefore the quantity of light exiting the samplesthat can be collected by the optics. According to various embodiments,the sample 10 can have an index of refraction 1.29 to 1.41, and thehousings 20 can have an index of refraction 1.46 to 1.6. According tovarious embodiments, an index matching fluid can be positioned betweenthe housing 20 and the NA enhancing optical element 40. According tovarious embodiments, air or other fluid can be positioned between thehousing 20 and the NA enhancing optical element 40.

According to various embodiments, the depth of sample along the opticalaxis can be small. According to various embodiments, the distance can be30 micrometers to 200 micrometers deep. The fluorescent light can beemitted in a narrow depth of field substantially decreasing sphericalaberrations. According to various embodiments, an aplanatic conditioncan be provided by positioning the sample at the radius of curvature ofa solid lens as described in patent application U.S. Ser. No. 09/564,790to Richard T. Reel titled “Optical System and Method for OpticallyAnalyzing Light from a Sample” that is herein incorporated by referencein its entirety. According to various embodiments, other aplanaticconditions are described in Kidger, Michael J., Fundamental OpticalDesign (2002), herein incorporated by reference in its entirety.

According to various embodiments, the amount of fluorescent lightcollected can be a function of the index of refraction (n₂) of the NAenhancing optical element 40 and the index of refraction (n₁) around theNA enhancing optical element 40. As illustrated in FIGS. 2A and 2B, NAenhancing optical element 40 can have radius (R) 60 and source plane 70where the source plane 70 has a distance 80 from the front end 90 of NAenhancing optical element 40, where the distance 80 can be calculated asR*(n₁+n₂)/n₂. The NA enhancing optical element 40 can refract thefluorescent light from source plane 70 so that it appears to come fromplane 100 and decrease the cone of light from half angle 120 to halfangle 130 providing more collection of fluorescent light. Plane 100 hasa distance 110 from the front end 90 of the NA enhancing optical element40, where the distance 100 can be calculated as R*(n₁+n₂)/n₁. Accordingto various embodiments, FIG. 2B illustrates a NA enhancing opticalelement 40 with a higher index of refraction than the NA enhancingoptical element 40 illustrated in FIG. 2A. Examples of high index ofrefraction materials include fused silica (n=1.46), optical glasses suchas BK7 (n=1.52) and LaSFN9 (n=1.85), and plastics such as polycarbonate(n=1.56). According to various embodiments, the material used toconstruct can be better served by using a low fluorescing material, lowRaleigh and low Raman scattering.

According to various embodiments, FIGS. 3A-3F illustrate different NAenhancing optical elements. FIG. 3A illustrates a NA enhancing opticalelement 40 including a truncated sphere 150 where the source plane 70can be the flat portion of the truncated sphere 150. FIG. 3B illustratesa NA enhancing optical element 40 including a sphere 160 combined with acylinder 170 where the source plane 70 can be the end of the cylinder170. FIG. 3C illustrates a NA enhancing optical element 40 including ameniscus lens 190 where the source plane 70 can be a plane at the radiusof curvature of the meniscus 200. FIG. 3D illustrates a NA enhancingoptical element 40 including a sphere 160 combined with a plate 180where the source plane 70 can be the end of the plate 180. FIG. 3Eillustrates a NA enhancing optical element 40 including a sphere 160combined with a fluid 140 and a plate 180. The sphere 160 can be bondedor coupled with the fluid 140 of similar index. The sphere 160 can bestationary or movable relative to plate 180 to provide scanning alongthe source plane 70.

According to various embodiments, the NA enhancing element and thehousing are constructed of different material. Unlike known systemswhere the index of refraction of a truncated sphere and a housing arematched so that spherical aberration can be eliminated, changing thematerial of the NA enhancing element according to the teachings of thepresent invention provides a significant increase in NA enhancement witha minimal introduction of spherical aberration. Unlike known systemswhere an index matching fluid is added to match both truncated sphereand housings and provide a continuum minimizing the refraction of theinterface of the truncated sphere and the housing, the index matchingfluid matches to either the truncated sphere, the housing, or anintermediate index.

According to various embodiments, as illustrated in FIG. 4, NA enhancingoptical element 40 can include truncated sphere 150 that can betruncated and/or positioned to provide an offset 205 between sourceplane 70 and the theoretical aplanatic source plane 70A. The offset 205can introduce corrective aberrations to fluorescent light detectionsystem when the truncated sphere 150 and housing 560 are constructed ofdifferent material, for example glass and fused silica. It is desirablethat the index of glass be used in the truncated sphere be as high aspractical. In a confocal configuration, where the NA enhancing opticalelement is used for excitation or collection, the improvementapproximately proportional to n⁴ each or n¹⁶ for both excitation andcollection. FIG. 4A illustrates truncating the sphere 150 andpositioning the housings 560 at the theoretical aplanatic source plane70A when the sphere 150 and the housings 560 are constructed ofdifferent material. The focus is shifted from the desirable position atthe center of the housing 560. FIG. 4B illustrates truncating the sphere150 and positioning the housings 560 at the source plane 70 that isoffset 205 from the theoretical aplanatic source plane 70A. The offset205 shift that focus to the desirable center of the housing 560 tocompensate for the difference in index of refraction of the truncatedsphere 150 and the housing 560.

According to various embodiments, as illustrated in FIG. 5, NA enhancingoptical element 40 can include truncated sphere 150 where the back end200 is curved to provide assistance in improving imaging across a curvedfield of view. According to various embodiments, additional opticalelements can be added to the NA enhancing optical element 40 illustratedin FIG. 5 to provide flattening of a curved field of view.

According to various embodiments, as illustrated in FIG. 6A,fluorescence light detection system 210 includes NA enhancing opticalelement 40 including truncated sphere 150 with offset source plane 70,lens 220, filter 240, lens 250, and detector 290. Lens 220 can form asubstantially collimated region 230 with fluorescent light 30 wherefilter 240 can be positioned to accept desired wavelengths of thefluorescent light 30 and reject other wavelengths. Filter 240 can be aninterference filter. Lens 250 can focus the fluorescent light 30 ontodetector 290. According to various embodiments, FIG. 6B illustrates afluorescent light detection system 210 similar to that illustrated inFIG. 6A except lens 260 focuses the fluorescent light 30 on filter 270that accepts desired wavelengths of the fluorescent light 30 and rejectsother wavelengths. Lens 280 focuses the fluorescent light 30 ontodetector 290. Filter 270 can be positioned on a filter wheel, linearactuator, or other mechanism for switching between multiple filters.

According to various embodiments, a fluorescent light detection systemcan include components to spectrally separate the wavelengths offluorescent light to provide multicolor detection including but notlimited to transmission gratings, reflective gratings, prisms, grisms,software controllable filters (width and position in spectral axis) etc.According to various embodiments, the sample container or thefluorescent light detection system can be positioned to decreasemotion-induced blurring on the detector. According to variousembodiments, a fluorescent light detection system can include foldingmirrors to decrease the size of the system.

According to various embodiments, as illustrated in FIG. 7, fluorescentlight detection system 210 can include light source 310, filter wheel300 including filter 220 and filter 370, mirrors 320 and 330, lenses340, 260, 360, and 280, NA enhancing optical element 40 including sourceplane 70, and detector 290. Excitation light 350 can be provided bylight source 310. Excitation light can be filtered by filter 220 onfilter wheel 300. Excitation light 350 can be directed to filter 370,such as a dichroic filter, on filter wheel 300 by mirrors 320 and 330and lens 340. The filter 370 reflects excitation light 350 towards lens260, NA enhancing optical element 40, and a sample at source plane 70.The large collection angle of NA enhancing optical element 40 canprovide an increased amount of light from light source 310 to a smalldetection spot of the sample. The excitation light 350 can be absorbedby the sample or dyes in the sample that can emit fluorescent light 30.Fluorescent light 30 can be collected by NA enhancing optical element 40and directed to lens 260 and filter 370. The color of fluorescent light30 can pass through filter 370 to lens 360 and lens 280 that focus thefluorescent light 30 onto detector 290. The image can be magnified toreduce the collection angle to one that is suitable for the detector290. According to various embodiments, lens 500 can be larger indiameter than lens 430 to avoid vignetting by making the angle ofincidence for the reflection of the dichroic mirror 480 be less than 45degrees to improve transmission and reflection properties.

According to various embodiments, as illustrated in FIG. 8, fluorescentlight detection system 210 can include two NA enhancing optical elements40 to collect fluorescent light from both sides of the sample. Thefluorescent light 30 on the opposite side of detector 290 can becollected and directed back towards detector 290 through lens 400 andmirror 410, such as a concave mirror. The fluorescent light 30 reflectedfrom mirror 410 can be directed back through both NA enhancing opticalelements 40 (co-imaged across source plane 70), lenses 220 and 250, andfilter 240 toward detector 290. Collecting light from both sides of thesample can double the amount of excitation light as well as doubling theamount of fluorescent light 30 directed toward the detector 290resulting in a 4× improvement. According to various embodiments, FIG. 9illustrates a fluorescent light detection system 210 similar to the oneillustrated in FIG. 7 with two NA enhancing optical elements 40 and amirror 410 as illustrated in FIG. 8.

According to various embodiments, a fluorescent light detection systemcan include a mask providing an aperture to reduce cross-talk betweenmultiple samples. According to various embodiments, the mask can bepositioned to provide excitation and collection of fluorescent lightfrom a single sample. According to various embodiments, samples can bepositioned so that the mask provides excitation light to and collectionof fluorescent light from a single sample. According to variousembodiments, the mask can be positioned to provide excitation andcollection of fluorescent light from a subset of samples. According tovarious embodiments, samples can be positioned so that the mask providesexcitation and collection of fluorescent light from a subset of samples.According to various embodiments, a fluorescent light detection systemcan be positioned to collect fluorescent light from each sample ondifferent portions of the detector thereby collecting individually whiledetecting collectively.

According to various embodiments, as illustrated in FIG. 10, fluorescentlight detection system 210 can include sample housings 560, NA enhancingoptical element 40, lenses 430, 460, 520, 500 and 800, dichroicbeam-splitter 480, light source 310, mask 440, grating 510 and detector290. According to various embodiments, mask 440 can be positioned toreduce cross-talk between multiple samples, as illustrated by the doublearrow near the mask. Lenses 800, which can be positioned on either sideof the mask as indicated by the broken lines, can focus light on mask440 while remaining stationary. According to various embodiments, lightsource 310 can be positioned in coordination with positioning mask 440,as illustrated by the double arrow near the light source. The lightsource can be narrow on the order of only one aperture in the mask.Hence, the light source can be positioned from aperture to aperture in amask by scanning with more than one aperture. According to variousembodiments, sample housings 560 and/or NA enhancing optical element 40can be positioned to reduce cross-talk between multiple samples, asillustrated by the double arrow near the sample housings. According tovarious embodiments, lens 500 and/or detector 290 can be positioned toreduce cross-talk between multiple samples, as illustrated by the doublearrow between the lens and detector. According to various embodiments, amirror can be positioned to direct where the image falls on thedetector. According to various embodiments, the detector can be maskedto control where the image falls on the detector.

According to various embodiments, as illustrated in FIG. 11A,fluorescent light detection system 210 can include assembly 550 that caninclude sample housings 560 in index matching fluid 420, NA enhancingoptical element 40, lenses 430, 500, 520, 460, and 800, dichroicbeam-splitter 480, filter 470, mirror 530, grating 510 and mask 440. Thelight source 310 can provide excitation light 250 to lens 460 that cancollect the excitation light 250 from the source. Excitation light 250can be directed to filter 470 that can condition the excitation light250 by accepting desirable wavelengths of excitation light 250 whileblocking other wavelengths. For example, filter 470 can be a bandpassfilter that accepts wavelengths that excite a dye in the sample andblock wavelengths that correspond to the wavelengths of the fluorescentlight emitted by the dyes. Excitation light 250 can be reflected bydichroic mirror 480 to pass through an aperture in mask 440. Mask 440can be positioned at an image plane of lens 430. The mask 440 cantranslate along the image plane as illustrated by the double arrow.Lenses 800 can focus light on mask 440 while remaining stationary.Excitation light 250 can be directed to NA enhancing optical element 40and focused onto housings 560 that can be positioned in source plane 70.Excitation light 250 can be absorbed by dyes in the samples in housings560, stimulating the dyes to emit fluorescent light 30 in alldirections. NA enhancing optical element 40 can collect fluorescentlight 30 and direct it to lens 430 and mask 440. The aperture in mask440 can bound excitation light 250 and fluorescent light 30 to onehousing 560 thereby reducing cross-talk between fluorescent lightemitted from other housings 560. Fluorescent light 30 can pass throughdichroic mirror 480, while non-fluorescent light can be rejected.Fluorescent light 30 can be reflected by mirror 530, substantiallycollimated by lens 520, dispersed by transmission grating 510, andfocused by lens 500 onto detector 290.

According to various embodiments, FIG. 11B illustrates a magnified viewof portion 540 of detector 290 illustrated in FIG. 11A. Portion 540 hasa spectral axis 570 and a spatial axis 580. Fluorescent light 30 from asingle housing 560 can be dispersed across band 595 of the detector.Regions of the detector, for example 590 and 600 collect differentwavelengths of light and can be measured to determine the spectralcomposition of the fluorescent light. Light from other housings 560dispersed across band 605 can be displaced on the detector so differentregions can be selected. The overlap of bands 595 and 605 can be reducedby clearing the detector after light from each housing is read.According to various embodiments, the region 590 can be 520 nanometersand region 600 can be 700 nanometers. According to various embodiments,fluorescent light can be collected from the detection zone while bandsof dye move through the housing. According to various embodiments,blurring induced by the motion of bands of dye can be reduced byshifting the packets of charges on the detector at the same velocity asthe image of the moving bands of dye on the detector. The velocity ofthe bands of dye passing the detection zone can be predicted because thebands of dye move at a predictable rate through the housing. Thevelocity of the bands of dye in the detection zone can be calculated bydividing the known separation distance by the measured separation time.According to various embodiments, the packets of charges on the detectorcan be accumulated by time-delay integration as described in U.S. Ser.No. 10/205,028 to Nordman et al. titled “Time-Delay Integration inElectrophoretic Detection Systems” that is herein incorporated byreference in its entirety. According to various embodiments, the maskcan be positioned to excite and/or detect a different housing and/orsubset of housings. According to various embodiments, the fluorescentlight detection system can cycle through all the housings and/or subsetsof housings such that the bands of dye detected at the beginning of thecycle have traveled no more than the full length of the detection zoneduring a cycle. Each cycle can capture a portion of the electropherogramfrom each housing. The spatial axis of the image of fluorescent bandscan be converted from distance to time to create a conventionalelectropherogram. According to various embodiments, the number ofhousings that can fit on the source plane for the collector lens candetermine the desirable cycling times/rate.

According to various embodiments, as illustrated in FIG. 12A,fluorescent light detection system 210 can include light source 310,lenses 460, 430, and 500, NA enhancing optical element 40, filter 470,grating 510, dichroic mirror 480, detector 290, housings 560, and mask440. The light source 310 can provide excitation light 250 to lens 460that can collect the excitation light 250 from the source. Excitationlight 250 can be directed to filter 470 that can condition theexcitation light 250 by accepting desirable wavelengths of excitationlight 250 while blocking other wavelengths. For example, filter 470 canbe a bandpass filter that accepts wavelengths that excite a dye in thesample and block wavelengths that correspond to the wavelengths of thefluorescent light emitted by the dyes. Excitation light 250 can bereflected by dichroic mirror 480 and directed to NA enhancing opticalelement 40 by lens 430 to be focused onto housings 560 through mask 440,where the housings 560 can be positioned in source plane 70 offset fromNA enhancing optical element 40. Excitation light 250 can be absorbed bydyes in the samples in housings 560, stimulating the dyes to emitfluorescent light 30 in all directions.

According to various embodiments, the fluorescent light detection systemillustrated in FIG. 12A can include a mechanism to translate housings560 in the source plane 70 so that mask 440 can align with one housingor a subset of the housings. This translation is illustrated in FIG. 12Cas a double-headed arrow near housing 560. According to variousembodiments, as illustrated in FIG. 12B, mask 440 can be coupled to theback end 200 of NA enhancing optical element 40 so that the aperture inmask 440 provides detection zone 630. NA enhancing optical element 40can collect fluorescent light 30 from the detection zone 630 and directit to lens 430. The detection zone 630 in mask 440 can bound excitationlight 250 and fluorescent light 30 to one housing or subset of housingsthereby reducing cross-talk between fluorescent light emitted from otherhousings 560. Fluorescent light 30 can pass through dichroic mirror 480,while non-fluorescent light can be rejected. Fluorescent light 30 can bedispersed by transmission grating 510, and focused by lens 500 ontodetector 290.

According to various embodiments, as illustrated in FIG. 12C, thehousings 560 can be positioned in assembly 550 so that the housings 560are immersed in index matching fluid 420. Assembly 550 can include NAenhancing, optical element holder 610 and base 650. Mask 440 can becoupled to back end 200 of NA enhancing optical element 40 so that theaperture in mask 440 provides detection zone 630. Mask 440 can be acoating applied to the back end 200 of NA enhancing optical element 40.According to various embodiments, the base 650 can be translated toposition housings 560 and index matching fluid 420 so that mask 440 canprovide detection zone 630 to the desired position to collectfluorescent light from one housing or a subset of housings. According tovarious embodiments, the housings 560 can be positioned in sequence toalign with the detection zone 630. According to various embodiments,base 650 can include baffling 640. Baffling 640 can form the bottom ofassembly 550. According to various embodiments, baffling 640 can includean anti-reflective window to permit the excitation light 250 and/orfluorescent light 30 to exit assembly 550 on the opposite side of NAenhancing optical element 40. For example, the window can be constructedof fused silica coated with an AR material to minimize background light.According to various embodiments, baffling 640 can include a mirror toreflect fluorescent light 30 and increase the amount of fluorescentlight 30 transmitted through NA enhancing optical element 40. Accordingto various embodiments, the mirror can be a spherical surface asillustrated in FIGS. 8 and 9.

According to various embodiments, FIG. 12D illustrates a magnified viewof portion 540 of detector 290 illustrated in FIG. 12A. Portion 540 hasa spectral axis 570 and a spatial axis 580. Fluorescent light 30 from asingle housing 560 can be spectrally separated into differentwavelengths, for example band 620. According to various embodiments,overlapping bands can be prevented by collecting fluorescent light 30from each housing 560 before switching to next housing 560. Theintegration time to collect light from each housing 560 can limit thenumber of housings 560. According to various embodiments, band 620 canrange from 520 nanometers and 700 nanometers. According to variousembodiments, the number of housings that can be positioned by the basecan be determined by at least one of the base translation time, detectorcollection time, size of detection zone, and sample rate. According tovarious embodiments, sample rate can include rate at which sampletravels through housing and/or rate of electrophoresis.

According to various embodiments, the fluorescent light detection systemillustrated in FIG. 12A can include a translation mechanism to move NAenhancing optical element 40. This translation is illustrated by thedouble-headed arrow near holder 610. According to various embodiments,as illustrated in FIGS. 12B and 12C and described herein, NA enhancingoptical element 40, mask 440, and assembly 550 can be similar to asystem that includes a translation mechanism to move the housings 560.Detection zone 630 can be positioned by the translation mechanism tomove the NA enhancing optical element 40. According to variousembodiments, FIG. 12E illustrates a magnified view of portion 540 ofdetector 290 illustrated in FIG. 12A. Portion 540 has a spectral axis570 and a spatial axis 580. Fluorescent light 30 can be detected atdifferent wavelengths from each housing 560, for example bands 660, 670,680, and 690, each from a different housing 560. According to variousembodiments, each band 660, 670, 680, and 690 can range from 520nanometers and 700 nanometers. According to various embodiments, thepitch or spacing of the housings 560 can be provided such that bands ofadjacent housings 560 do not overlap, as illustrated in FIG. 12E. Thewavelength bands for each housing 560 can be detected on the detector290 at one interval without having to be detected separately. Accordingto various embodiments, the detector can accumulate packets of chargesby time-delay integration. The detector collection time can be matchedto the sample rate.

According to various embodiments, gaps 700 can provide a range ofwavelengths to permit bandpass filter 470 to exclude sufficientwavelengths to reduce overlap between bands 660, 670, 680, and 690 onportion 540 of detector 290. According to various embodiments, pitch, P,can be calculated: P=S*R/M, wherein S is the number of pixels ondetector 290 to capture one of bands and a gap, R is the width perpixel, and M is the magnification. According to various embodiments, thesize of the detector 290 can determine the number of housings 560detected at the same time.

According to various embodiments, as illustrate in FIG. 13, chromaticaberrations can be reduced by tilting the detector 290 and positioninghousings 560 such that source plane 70 tilts. For example, tiltingsource plane 70 to position 70A as illustrated by the arrow and tiltingthe plane 710 of detector 290 to position 710A provides detection offluorescent light 30 collected from housing 560A to be detected bywavelength band 720A and fluorescent light 30 collected from housing560B to be detected by wavelength band 720B. According to variousembodiments, a fluorescent light detection system including the tiltingillustrated in FIG. 13, can reduce chromatic aberrations by distancingdifferent colors.

According to various embodiments, the fluorescent light detection systemillustrated in FIG. 12A can include a mechanism to translate mask 440.This translation is illustrated by the double-headed arrow near mask 440in FIG. 12C. According to various embodiments, as illustrated in FIGS.12B and 12C and described herein, NA enhancing optical element 40, mask440, and assembly 550 can be similar to system that includes atranslation mechanism to move the housings 560 except that the mask 440is not coupled to NA enhancing optical element 40. Detection zone 630can be positioned by the mechanism to translate the mask 440. Accordingto various embodiments, as illustrated in FIG. 12D and described herein,the fluorescent light detection system can provide band 620 to a portion540 of detector 290.

According to various embodiments, the fluorescent light detection systemcan collect fluorescent light from a subset of housings. According tovarious embodiments, the mask can include two or more apertures toprovide multiple detection zones. According to various embodiments, theapertures can be aligned such that fluorescent light can be collectedfrom non-adjacent housings. This can provide collection of fluorescentlight from multiple housings. According to various embodiments, asillustrated in FIGS. 14 and 14A, mask 440 can include multiple detectionzones 630. According to various embodiments, as illustrated in FIG. 14B,portion 540 of detector 290 can detect fluorescent light collected froma first detection zone as wavelength band 730 and from a seconddetection zone as wavelength band 740 without overlap. According tovarious embodiments, the fluorescent light detection system can includea dichroic mirror that provides bandpass filtering to reject wavelengthsoutside the range of interest to reduce overlap of wavelength bands.

According to various embodiments, as illustrated in FIG. 15, mask 440can include multiple apertures to provide detection zones 630 to collectfluorescent light 30 from non-adjacent housings 560. According tovarious embodiments, as illustrated in FIG. 16, mask 440 can include twoapertures to provide detection zones 630 to collect fluorescent light 30from non-adjacent housings 560. Housings 560 can be tilted from sourceplane 70 to source plane 71 to compensate for tilting detector 290 toreduce chromatic aberrations. According to various embodiments, thefluorescent light can be collected from the remaining housings by atleast one of: (1) translating the housings 560; (2) translating the NAenhancing optical element 40 coupled to the mask 440; (3) translatingthe mask not coupled to the NA enhancing optical element 40 wherein themask can be positioned between the housings 560 and NA enhancing opticalelement 40; and (4) translating the mask not coupled to the NA enhancingoptical element 40 wherein the mask can be positioned between NAenhancing optical element 40 and detector 290.

According to various embodiments, the fluorescent light detection systemcan be included in an instrument for detection of fluorescent light fromelectrophoresis. According to various embodiments, the fluorescent lightdetection system can be included in an instrument for detection offluorescent light from flow cytometry. According to various embodiments,the fluorescent light detection system can be included in an instrumentfor detection of fluorescent light from liquid chromatography, such ashigh-pressure liquid chromatography (HPLC).

According to various embodiments, a method for fluorescent lightdetection can include providing a plurality of housings for the samples,providing a NA enhancing optical element, providing a mask, andpositioning the mask to reduce cross-talk between fluorescent light fromthe samples. According to various embodiments, positioning the mask caninclude positioning the mask between the NA enhancing optical elementand the plurality of housings. According to various embodiments,positioning the mask further include translating the plurality ofhousings. According to various embodiments, positioning the mask caninclude translating the NA enhancing optical element and the mask.According to various embodiments, positioning the mask can includetranslating the mask. According to various embodiments, positioning themask can include positioning the mask between the NA enhancing opticalelement and a detector.

All publications and patent applications referred to herein are herebyincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

For the purposes of this specification and appended claims, unlessotherwise indicated, all numbers expressing quantities, percentages orproportions, and other numerical values used in the specification andclaims, are to be understood as being modified in all instances by theterm “about.” Accordingly, unless indicated to the contrary, thenumerical parameters set forth in the following specification andattached claims are approximations that may vary depending upon thedesired properties sought to be obtained by the present invention. Atthe very least, and not as an attempt to limit the application of thedoctrine of equivalents to the scope of the claims, each numericalparameter should at least be construed in light of the number ofreported significant digits and by applying ordinary roundingtechniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Moreover, all ranges disclosed hereinare to be understood to encompass any and all subranges subsumedtherein. For example, a range of “less than 10” includes any and allsubranges between (and including) the minimum value of zero and themaximum value of 10, that is, any and all subranges having a minimumvalue of equal to or greater than zero and a maximum value of equal toor less than 10, e.g., 1 to 5.

It is noted that, as used in this specification and the appended claims,the singular forms “a,” “an,” and “the,” include plural referents unlessexpressly and unequivocally limited to one referent. Thus, for example,reference to “a mask” includes two or more different masks. As usedherein, the term “include” and its grammatical variants are intended tobe non-limiting, such that recitation of items in a list is not to theexclusion of other like items that can be substituted or added to thelisted items.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to various embodimentsdescribed herein without departing from the spirit or scope of thepresent teachings. Thus, it is intended that the various embodimentsdescribed herein cover other modifications and variations within thescope of the appended claims and their equivalents.

1. A fluorescent light detection system for analyzing biological samplescomprising: at least one NA enhancing optical element; and at least onehousing for the samples, wherein the NA enhancing optical element isconstructed of a first material and the housing is constructed of asecond material, wherein the first material has a greater index ofrefraction than the second material.
 2. The system of claim 1, whereinthe samples being detected are located in a source plane offset from thetheoretical aplanatic source plane of the NA enhancing optical element.3. The system of claim 2, wherein the offset is configured to providecorrective aberration.
 4. The system of claim 2, wherein the NAenhancing optical element is a truncated sphere.
 5. The system of claim4, wherein the truncated sphere is truncated such that the source planesubstantially intersects the center of the housings.
 6. The system ofclaim 1, further comprising an index matching fluid.
 7. The system ofclaim 6, wherein the index matching fluid has an index of refractionsubstantially equal to the first material.
 8. The system of claim 6,wherein the index matching fluid has an index of refractionsubstantially equal to the second material.
 9. A fluorescent lightdetection system for analyzing biological samples comprising: a NAenhancing optical element; a plurality of housings for the samples; amask comprising at least one aperture adapted to reduce cross-talk offluorescent light from the samples; and a translation mechanism, whereinthe translation mechanism is adapted to move at least one of the NAenhancing optical element, the plurality of housings, and the mask. 10.The system of claim 9, wherein the NA enhancing optical element is atruncated sphere.
 11. The system of claim 10, wherein the mask ispositioned between a back end of the truncated sphere and the pluralityof housings.
 12. The system of claim 11, wherein the mask is coupled tothe back end of the truncated sphere.
 13. The system of claim 10,wherein the mask is positioned between a front end of the truncatedsphere and the detector.
 14. The system of claim 10, further comprisinga detector adapted to detect fluorescent light from multiple housingsper cycle.
 15. The system of claim 10, wherein the detector providestime-delay integration.
 16. The system of claim 10, wherein the detectoris tilted to reduce chromatic aberration.
 17. The system of claim 10,further comprising a detector adapted to detect fluorescent light fromone housing per cycle.
 18. The system of claim 10, wherein thetranslation mechanism provides translation of a detection zone, whereinthe translation substantially matches the speed of the samples in thehousings.
 19. The system of claim 10, wherein the mask comprisesmultiple apertures adapted to correspond to non-adjacent housings. 20.The system of claim 10, further comprising a grating adapted to providefluorescent light from multiple dyes to a detector.
 21. A fluorescentlight detection method for analyzing biological samples comprising:enhancing the NA of the system with a NA enhancing optical element; andreducing cross-talk between fluorescent light from samples in aplurality of housings.
 22. The method of claim 21, wherein enhancing NAcomprises truncating a spherical lens, and coupling the truncated sphereto at least one housing, wherein the spherical lens is constructed of afirst material and the housing is constructed of a second material, suchthat the first material has a greater index of refraction than thesecond material.
 23. The method of claim 22, wherein reducing cross-talkcomprises positioning a mask between the truncated sphere and theplurality of housings.
 24. The method of claim 23, wherein positioningthe mask further comprises translating the plurality of housings. 25.The method of claim 23, wherein positioning the mask further comprisestranslating the truncated sphere and the mask.
 26. The method of claim23, wherein positioning the mask further comprises translating the mask.27. The method of claim 22, reducing cross-talk comprises positioning amask between the truncated sphere and a detector.
 28. The method ofclaim 27, wherein positioning the mask further comprises translating themask.
 29. A fluorescent light detection system for analyzing samplescomprising: means for enhancing NA; means for housing the samples; andmeans for reducing cross-talk of fluorescent light from samples inmultiple means for housing.
 30. The system of claim 29, furthercomprising means for correcting aberration.
 31. A fluorescent lightdetection system for analyzing biological samples comprising: at leastone NA enhancing optical element; and at least one housing for thesamples, wherein the NA enhancing optical element is constructed of afirst material and the housing is constructed of a second material,wherein the first material has a greater index of refraction than thesecond material, and wherein the NA enhancing optical element providesexcitation light to induce the fluorescent light on a single opticalpath between the NA enhancing optical element and the samples.